Open AccessNew technique for high resolution DNA sizing in epi‐illumination
Author(s) -
Schins J. M.,
Agronskaya A.,
de Grooth B. G.,
Greve J.
Publication year - 1998
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19980601)32:2<132::aid-cyto8>3.0.co;2-n
Subject(s) - sizing , resolution (logic) , particle (ecology) , fluorescence , detection limit , numerical aperture , dna , high resolution , flow cytometry , cytometry , limit (mathematics) , materials science , optics , flow (mathematics) , biological system , computer science , chromatography , chemistry , physics , optoelectronics , biology , remote sensing , microbiology and biotechnology , mathematics , geology , artificial intelligence , wavelength , genetics , ecology , mathematical analysis , mechanics , organic chemistry
We present a high‐resolution DNA‐sizing technique based on the principles of flow cytometry, using a high numerical aperture objective and epi‐illumination. The new technique, designed for small fluorescing samples/particles (sub‐micron diameter) suspended in a weakly fluorescent medium, makes use of an additional focus for high‐precision particle localisation. This way, only those particles are considered that flow exactly through a well‐defined volume. Results are presented for fluorescent beads, as well as for YOYO‐stained plasmids containing 5,500 basepairs. The latter were measured with 6.2% resolution, setting a new limit to flow‐based sizing of DNA. Cytometry 32:132–136, 1998. © 1998 Wiley‐Liss, Inc.